1. Field of the Invention
The present invention relates to the FokI restriction endonuclease system. In particular, the present invention relates to DNA segments encoding the separate functional domains of this restriction endonuclease system.
The present invention also relates to the construction of two insertion mutants of FokI endonuclease.
Additionally, the present invention relates to a hybrid enzyme (Ubx-F.sub.N) prepared by linking the Ultrabithorax Ubx homeo domain to the cleavage domain (F.sub.N) of FokI.
2. Background Information
Type II endonucleases and modification methylases are bacterial enzymes that recognize specific sequences in duplex DNA. The endonuclease cleaves the DNA while the methylases methylate adenine or cytosine residues so as to protect the host-genome against cleavage [Type II restriction and modification enzymes. In Nucleases (Eds. Modrich and Roberts) Cold Spring Harbor Laboratory, New York, pp. 109-154, 1982]. These restriction-modification (R-M) systems function to protect cells from infection by phage and plasmid molecules that would otherwise destroy them.
As many as 2500 restriction enzymes with over 200 specificities have been detected and purified (Wilson and Murray, Annu. Rev. Genet. 25:585-627, 1991). The recognition sites of most of these enzymes are 4-6 base pairs long. The small size of the recognition sites is beneficial as the phage genomes are usually small and these small recognition sites occur more frequently in the phage.
Eighty different R-M systems belonging to the Type IIS class with over 35 specificities have been identified. This class is unique in that the cleavage site of the enzyme is separate from the recognition sequence. Usually the distance between the recognition site and the cleavage site is quite precise (Szybalski et al., Gene, 100:13-26, 1991). Among all these enzymes, the FokI restriction endonuclease is the most well characterized member of the Type IIS class. The FokI endonuclease (RFokI) recognizes asymmetric pentanucleotides in double-stranded DNA, 5' GGATG-3' (SEQ ID NO: 1) in one strand and 3'-CCTAC-5' (SEQ ID NO: 2) in the other, and introduces staggered cleavages at sites away from the recognition site (Sugisaki et al., Gene 16:73-78; 1981). In contrast, the FokI methylase (MFokI) modifies DNA thereby rendering the DNA resistant to digestion by FokI endonuclease. The FokI restriction and modification genes have been cloned and their nucleotide sequences deduced (Kita et al., J. of Biol. Chem., 264:575-5756, 1989). Nevertheless, the domain structure of the FokI restriction endonuclease remains unknown, although a three domain structure has been suggested (Wilson and Murray, Annu. Rev. Genet. 25:585-627, 1991).